Abstract

CD99 is a transmembrane 32 KDa protein, whose expression is constantly associated to Ewing's Sarcomas Family of Tumors (EWSFT), a class of child bone tumors with particular poor prognosis. The monoclonal antibody 0662, raised against CD99, triggers massive and rapid cell death of Ewing Sarcoma's cells in vitro and in vivo, through a non-canonical, non caspase-dependent apoptosis. To elucidate the underlying molecular mechanism, we evaluated by phosphor- and micro-arrays specific pathways triggered by CD99 engagement in 6647 Ewing Sarcoma cell line. Despite its role in physiological and cancer scenery has not been yet fully elucidated, CD99 appears to be sufficient to modulate many important processes involved in: I) adhesion; II) migration and metastasis; III) cell-cycle regulation and cell signaling; IV) apoptosis and death receptor signaling. These data are consistent with our previous analysis of 0662 effects in vivo and in vitro: anti-CD99 monoclonal antibody triggers, indeed, homotypic aggregation and a rapid death response of ES cells, thus resulting in growth inhibition and reduced metastatic/colony-forming potential. As pointed out by array studies and subsequent western blotting validations, upon 0662 treatment, the oncosuppressor protein p53 appears to be readily activated by phosphorilation on serine 15. We therefore evaluated p53 and MDM2 activation and sub-cellular localization upon CD99 engagement both in p53 WT (LAP35 and WE68) or mutated (6647 and TC-71) cell lines: WT (LAP35 and WE68) or mis-sense point mutated p53 (6647: S241F) cell lines display a higher sensitivity to 0662 if compared to p53 truncated cell lines (TC71). Consistently we demonstrated that whereas 0662 readily induces p53 canonical targets (p21, BAX) in p53 expressing cell lines, no increase in p21 levels is detectable in TC71 p53TRUNC cells. We also demonstrated that, upon 0662 treatment, decreased or delocalized MDM2 levels may account both for p53 activation and sustained IGF-IR signaling, since both p53 and IGF1-Rß are targeted by the ubiquitine ligase for degradation. Since most Ewing Sarcoma patients display unmodified p53 status, its functional inactivation -e.g. through MDM2/MDM4 overexpression- may account for gained tumor aggressiveness, or progressed disease. Nonetheless, involvement of TP53 might give reason for higher effectiveness we reported against local tumors and metastases in the combined employment of CD99 monoclonal antibodies with Doxorubicin and may support 0662 mAb treatment in association with other drugs targeting e.g. the p53 regulators MDM2-MDM4. (grants from EU project Eurobonet and Italian Association for Cancer Research)